DNA damage is an integral component of hereditary diseases and aging. The overall goals of the proposed research are to examine fundamental questions concerning how DNA is damaged at the molecular level and to apply the knowledge gained from these studies to the design of therapeutic agents. Consequently, this research is valuable to understanding the etiology and treatment of diseases such as cancer, and provides insight into how we age. These studies are accomplished using a conglomeration of synthetic and physical organic chemistry along with techniques borrowed from molecular and radiation biology. Our general experimental approach invokes designing and synthesizing molecules that enable us to unambiguously generate reactive intermediates at defined sites in oligonucleotides. Using this approach, we can identify novel pathways of DNA damage, resolve mechanistic controversies, and probe the mechanism of action drugs and repair enzymes that interact with DNA. Specific aims from the current funding period include: 1. Design of mechanism-based radiosensitizing agents. 2. Determine the roles that reactive intermediates that may not be identified by damaged nucleotides after the fact ("invisible intermediate") play in DNA damage 3. Determine the ability of reactive species to amplify DNA damage by forming tandem and clustered lesions. 4. Determine the propensity for an oxidized nucleotide to catalyze formation of a premutagenic lesion.